Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post-translational modification. In this study, bioreactor production of recombinant human alpha-1-antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25-fold increase in extracellular functional rAAT (603 mu g/L) and a higher ratio of functional rAAT to total rAAT (85-90%). Surprisingly, sustained rAAT production and steady state, long-term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010;106: 408-421. (C) 2010 Wiley Periodicals, Inc.